Figure 7.
PDC1 and ENO2 mRNAs Are Translated in the Same mRNA Granules
(A) Epifluorescent microscopic z stack images of exponential cells expressing endogenous PP7L-tagged ENO2 (visualized via PP7-GFP2, left images) and either MS2L-tagged PDC1 or MS2L-tagged TIF1 (visualized via coexpressed MS2-mCherry3, middle images). Merged images are shown (right) with the percentage GFP granules overlapping with mCherry granules quantified across 50 cells for ENO2 v PDC1. No colocalization was observed for ENO2 v TIF1. Error bar is ±SE.
(B) Figure shows a FRAP experiment on the Eno2p-mOrange bearing strain yMK1993 where recovery after photobleaching is followed relative to the localization of the PDC1-MS2L mRNA (visualized using MS2-GFP3). Prebleach, bleached, and recovery images are shown for PDC1 mRNA (top row) and mOrange-tagged Eno2p protein (bottom row).
(C) Immunofluoresence using an antipuromycin antibody on cells treated with puromycin/cycloheximide to trap puromycin at the site of protein synthesis (center panels). The MS2-GFP3 mRNA signal for PDC1 and ENO2 is maintained during the procedure (left panels). Merged images show the overlap of the puromycin signal with the mRNA granules (right panels).
(D) As in (C), except puromycin was omitted from the procedure.
Scale bars, 2 μm throughout. See also Figures S5 and S6.